Amyloidogenic diseases such as Alzheimer's disease (AD) have been recognized as the major cause of dementia in elderly people. The decline of cognitive abilities in AD is associated with histopathological changes in the brain, the most relevant being the formation of amyloid plaques and neurofibrillary tangles.
While amyloid plaques contain many proteins, they have as their main constituent the amyloid-β (Aβ) peptide. The formation of the Aβ peptide, and thereby Aβ amyloid plaques, arises from aberrant processing of the amyloid precursor protein (APP).
Currently, several pharmacological approaches have being developed to slow or reverse the progression of AD. While several approaches are directed to inhibit the metabolic generation of the Aβ peptide, others are directed to prevent the aggregation of the Aβ amyloid in the brain of AD affected patients.
However, the most promising approaches are directed to increase the brain clearance of Aβ plaques through the administration of either antigens able to generate an immune response against Aβ (active immunization) or antibodies directed against Aβ (passive immunization).
Antigens or immunogens are usually macromolecules that contain distinct antigenic sites or “epitopes” that are recognized and interact with the various components of the immune system. They usually comprise a small molecule or “hapten”, such as short peptide, coupled to a suitable carrier. Carriers typically are proteins of higher molecular weight that are able to cause an immunological response when administered in vivo.
In an immune response, antibodies are produced and secreted by the B-lymphocytes in conjunction with the T-helper (TH) cells. In the majority of hapten-carrier systems, the B cells produce antibodies that are specific for both the hapten and the carrier. In these cases, the T lymphocytes will have specific binding domains on the carrier, but will not recognize the hapten alone. In a kind of synergism, the B and T cells cooperate to induce a hapten-specific antibody response.
Therefore, in constructing an effective antigen, the selection of the proper carrier and the proper hapten is crucial to guarantee a robust and selective immunogenic response. The safety of the antigen is also of crucial importance. For example, the administration to AD patients of the promising AN-1792 vaccine constituted by pre-aggregated Aβ42 and the immune adjuvant QS-21 led to severe meningoencephalitis in about 6% of the treated subjects. Both central activation of cytotoxic T cells and autoimmune reactions were proposed as potential mechanisms of toxicity. An immunological response against endogenous monomeric Aβ may be harmful since non-aggregated Aβ species have a physiological role in neuronal activity.
Thus, it is of great importance the proper selection of both the hapten and the carrier to guarantee antibody selectivity towards the harmful Aβ species and to prevent autoimmune toxicity.
WO2005058940 proposes conjugating peptide immunogen comprising Aβ peptide or a fragment thereof to a protein/polypeptide carrier.
The immunogenic constructs are produced by a chemical method comprising derivatizing functional groups of amino acid residues of the carrier wherein any unconjugated, derivatized functional groups of the amino acid residues are inactivated via capping to block them from reacting with other molecules. Such a method results in immunogens wherein the Aβ fragment is bound to the amino acid side chains of the carrier. While in WO2005058940 several different carriers and haptens have been proposed their in vivo histopathological efficacy has not been shown.
Kim, H. D. et al in Biochem. Biophys, Res. Commun. Volume 336, pages 84-92 propose an anti-Aβ DNA vaccine, composed of unscaffolded 11-fold repeats of Aβ1-6.
Such construct yielded antibodies that indiscriminately recognized monomeric, oligomeric and fibrillar Aβ42 species.
In general, selective targeting of immunogens against the different assembly states of Aβ42 (monomers, oligomers or fibrils) has not been achieved so far.
In view of the above considerations there is still a need to develop a safe and effective immunogenic construct which may be used in therapeuthic vaccination compositions to prevent the aggregation of Aβ amyloid in the brain of patients affected by AD or other amyloidogenic deseases such as Down Syndrome.
The present invention provides a recombinant immunogenic construct characterized in that the Aβ fragments is positioned within the active loop site (display site) of the carrier rather than bound to the ends of the carrier. Said peptide is obtained by tandem multimerization of a B-cell epitope bearing fragment of Aβ42, within the active loop site (display site) of a carrier, preferably thioredoxin (Trx).
The immunogens of the present invention were found to elicit antibodies recognizing neurotoxic oligomeric species of the Aβ amyloid which recently have been indicated as the most proximate causative agents of amyloidogenic diseases.
This capability has been associated with the construction of the immunogen featuring the Aβ amyloid within the carrier. Such configuration to some extent permits the right folding of the immunogenic protein and more effectively presents it to the immune system. When the immunogen bears more than one Aβ amyloid fragment, and in particular specific numbers of said fragments, the resemblance of the immunogen to the Aβ amyloid oligomers, is believed to further improve its efficacy as well as to increase the selectivity.
A linker between the carrier and the fragments further helps in preserving the peptide epitope assembly state.